Significantly Improved Expression and Biochemical Properties of Recombinant Serratia marcescens Lipase as Robust Biocatalyst for Kinetic Resolution of Chiral Ester

Abstract

A lipase gene from Serratia marcescens ECU1010 was cloned into expression vector pET28a, sequenced, and overexpressed as an N terminus His-tag fusion protein in Escherichia coli. Through the optimization of culture conditions in shake flask, the lipase activity was improved up to 1.09 x 10⁵ U/l, which is a great improvement compared to our previous reports. It was purified to homogeneity by Ni-NTA affinity chromatography with an overall yield of 59.4% and a purification factor of 2.4-fold. This recombinant lipase displayed excellent stability below 30 °C and within the pH range of 5.0-6.8, giving temperature and pH optima at 40 °C and pH 9.0, respectively. The lipase activity was found to increase in the presence of metal ions such as Ca²+, Cu²+, and some nonionic surfactants such as PEG series. In addition, among p-nitrophenyl esters of fatty acids with varied chain length, the recombinant lipase showed the maximum activity on p-nitrophenyl laurate (C₁₂). Using racemic trans-3-(4'-methoxy-phenyl)-glycidyl methyl ester [(±)-MPGM] as substrate, which is a key chiral synthon for production of diltiazem, a 50% conversion yield was achieved after 4 h in toluene-water (100 mM KPB phosphate buffer, pH 7.5) biphasic system (5:5 ml) at 30 °C under shaking condition (160 rpm), affording (-)-MPGM in nearly 100% ee. The K(m) and V(max) values of the lipase for (±)-MPGM were 222 mM and 1.24 mmol min⁻¹ mg⁻¹, respectively. The above-mentioned features make the highly enantioselective lipase from Serratia marcescens ECU1010 a robust biocatalyst for practical use in large-scale production of diltiazem intermediate.

Expression and Characterization of a Novel Lipase from Aspergillus fumigatus with High Specific Activity

“…The procedure for assay of lipase activity has been described previously [9]. The activity measurement was carried out at 30°C, pH 7.5 (100 mM KPB).One unit of lipase activity was defined as the amount of enzyme releasing 1.0 μmol of p-nitrophenol per minute under the assay conditions [10].…”

“…The recombinant plasmid was transformed into E. coli BL21 (DE3) for protein expression. Many physical and chemical factors such as IPTG concentration, incubation time, and temperature may affect the production and correct folding of the protein overexpressed in E. coli [9]; therefore, the induction conditions were optimized for enhancing the soluble expression of the recombinant enzyme. In order to collect massive soluble protein, 15°C was chosen for induction to avoid amount of inclusion body being produced at 37°C (data not shown).…”

“…Furthermore, the activity of AFL1-1 was almost independent of EDTA concentration within a certain range (Table 2). It was suggested that this recombinant lipase did not belong to metalloenzyme as those lipases from Serratia marcescens, Bacillus licheniformis, Mucor sp., and Bacillus thermoleovorans [9,[18][19][20]. Surfactants have been widely applied to lipase-catalyzed reactions of insoluble substrates to increase the lipid-water interfacial area, which in turn enhance the reaction rate of the kinetic resolution [21].…”

Kinetic modeling, production and characterization of an acidic lipase produced by Enterococcus durans NCIM5427 from fish waste

“…Lipase activity was determined spectrophotometrically as described by Wang et al (2010) using p-NPA as the substrate with slight modifications. LCFE (300 μl) was mixed with 900 μl solvent mixture (acetonitrile: ethanol: phosphate buffer (pH 6.8) in the ratio of 1:4:95 v/v/v) followed by the addition of 800 μl of 100 mM p-NPA in acetonitrile and incubated at 37°C for 15 min.…”

Expression and Characterization of a Novel Enantioselective Lipase from Aspergillus fumigatus

“…Lipase activity was examined by the spectrometric method using 100 mM 4-nitrophenyl hexanoate (p-NP hexanoate) as a substrate [25]. One unit of lipase activity was defined as the amount of enzyme releasing 1.0 μmol of p-nitrophenol per minute under the condition of 30°C and pH 7.0 (100 mM KPB) [26].…”